Abstract In developed countries, blood with rare phenotypes for alloimmunized patients is often unavailable. The use of cultured RBCs (cRBCs) to address these clinical needs gained momentum when our laboratory, among others, developed conditions for large scale production of cRBCs. Presently, several laboratories, including ours, are addressing the numerous barriers to establish cRBCs as clinical product. In 2011, the Douay laboratory demonstrated that 2.5x1010 autologous cRBCs (5 mL of blood) generated from mobilized CD34pos cells have normal survival in vivo. However, this ground breaking study has four caveats: 1) mobilization is unlikely to be accepted as procedure to generate cRBCs for clinical or diagnostic use, 2) the amount of cRBCs generated (~5 mL of blood) is one-log lower than the minimal dose required to transfuse a child (50 mL or 2.5x1011 RBCs), 3) normal volunteers are not ideal recipients for the first clinical trial to assess efficacy, and 4) the high costs to produce the cRBC limit their clinical use as product. This proposal will address these caveats by providing proof-of-principle that clinically useful doses of cRBCs can be generated from discarded stem cell sources and used for transfusion of alloimmunized patients with rare phenotypes for whom, having no alternatives, the costs to produce cRBCs is considered reasonable. This proposal is based on a close collaboration among basic scientists (Drs. Migliaccio and Bieker, Icahn School of Medicine and Mount Sinai) and clinicians (Drs. Klein and Flegel, Transfusion Medicine Department of the NIH). These investigators will perform experiments aimed to establish whether leukoreduction by- products currently discarded from regular blood donations with rare phenotypes are suitable to generate 2.5x1011 cRBCs (Aim 1) using novel culture strategies based on ?gene editing? that will increase yields while reducing production costs (Aim 2). We believe that the documented previous colloboration between Drs Migliaccio and Bieker, as well as the two new collaborators (Drs. Funnel and Zon), on scientific questions related to this application and the long standing interest of Drs Klein and Flegel in clinical management of Sickle Cell Anemia patients, including those with rare phenotypes, assures the synergistic development of scientific and clinical aspects of this proposal. This is necessary to maximize the likelihood of efficiently producing adequate numbers of cRBCs well-suited for the first-in-man allogenic transfusion.